Het effect van dejodase-deficiëntie op de ontwikkeling en voortplanting van de zebravis

Thyroid hormones (THs) are crucial regulators for a wide range of processes in the body. In this thesis we focus on development, growth and reproduction, which are all tightly controlled by TH action. The thyroid gland mainly produces the prohormone 3,5,3’,5’-tetraiodothyronine (T4) and to a much lesser extent the receptor-active 3,5,3’-triiodothyronine (T3). Appropriate T3 availability is locally regulated by deiodinases (Dio1, Dio2, Dio3), the enzymes that activate and inactivate THs. To unravel the role of deiodinases during these essential processes, we used knockdown and knockout technologies in the zebrafish model. Based on a transcriptome analysis performed in deiodinase knockdown embryos, where we observed a strong downregulation of genes related to phototransduction, eye development and visual perception, we carried out in-depth analysis of the eye phenotype at the level of gene transcription, morphology and function. The expression of all components of the phototransduction and retinoid recycling pathway was adversely affected. This was accompanied by decreased eye size, disturbed retinal lamination and strong reduction in rods and all 4 types of cone photoreceptors. These defects were more prominent and persistent in dio3-deficient fish, while the Dio1+Dio2 knockdown phenotype seemed to reflect primarily a general delay in early development. Dio3-deficient larvae in addition had disrupted visual function and were less sensitive to a light stimulus. Since morpholinos only induce a transient knockdown, we used the zinc finger nuclease technology to generate the first non-mammalian model with permanent deiodinase deficiency, thereby focusing on Dio2, the main TH activator in zebrafish. Two different dio2-/- mutant lines were generated: the deletion and insertion mutant. Both lines completely and specifically lack Dio2 activity, resulting in a strong reduction in T3 abundancy in all tissues tested. Together with the inability of Dio3 to compensate for these diminished T3 levels, TR expression is downregulated, pointing to a decreased TH signaling in our mutant fish. Early development was clearly perturbed in these animals, as shown by a diverse set of morphometric parameters, defects in swim bladder inflation and disturbed locomotor activity until 7 days of age. Long-term defects were also found in the growth of the fish, especially in males. The most striking long-term defect was observed in reproduction. dio2-/- mutant fish started reproducing at a later age than WT fish and their reproductive period was severely shortened. In addition, females laid significantly less eggs and males displayed fertilization problems. More profound analysis of gametogenesis did not reveal major problems in female oogenesis, but however pointed in the direction of ovulatory disturbances. Spermatogenesis in males was more strongly influenced, with indications of ejaculatory dysfunction or possibly sperm quality. The search for the underlying molecular mechanisms exposed downregulation of gametogenesis- and steroidogenesis-related genes, which was more prominent in males compared to females. A first series of experiments also suggested courtship behavior issues, which may be correlated to impaired/altered pheromone production in one or both sexes, that possibly led to diminished male typical behavior. Taken together, these data show that an accurate balance between TH activation and inactivation is indispensable for development, growth and reproduction to proceed normally. Importantly, we have also demonstrated that zebrafish is an excellent model to further explore the role of deiodinases. The newly generated dio2-/- mutant lines will definitely help in future studies to increase our understanding of vertebrate TH metabolism and its implications for human health.status: publishe